The AHA Clinical SeriesSERIES EDITOR ELLIOTT ANTMAN

Antiplatelet Therapy In Ischemic Heart Disease

To my family Caroline, Jack, Sam and Lucy

Antiplatelet Therapy In Ischemic Heart DiseaseEDITED BY

Stephen D. Wiviott, MDCardiovascular DivisionBrigham and Women’s HospitalAssistant Professor of MedicineHarvard Medical SchoolInvestigator, TIMI Study GroupBoston, MA

SERIES EDITOR ELLIOTT ANTMAN

The AHA Clinical Series

A John Wiley & Sons, Ltd., Publication

This edition fi rst published 2009, © 2009 American Heart AssociationAmerican Heart Association National Center, 7272 Greenville Avenue, Dallas, TX 75231, USAFor further information on the American Heart Association:www.americanheart.org

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Library of Congress Cataloging-in-Publication DataAntiplatelet therapy in ischemic heart disease / edited by Stephen D. Wiviott. p. ; cm.—(AHA clinical series) Includes bibliographical references. ISBN 978-1-4051-7626-2 1. Coronary heart disease—Chemotherapy. 2. Blood platelets—Aggregation. I. Wiviott, Stephen D. II. American Heart Association. III. Series. [DNLM: 1. Myocardial Ischemia—drug therapy. 2. Platelet Aggregation Inhibitors—therapeutic use. 3. Myocardial Ischemia—blood. QV 180 A634 2009] RC685.C6A69 2009 616.1’23061—dc22 2008037317

ISBN: 9781405176262A catalogue record for this book is available from the British Library.

Set in Palatino 9/12 by Charon Tec Ltd (A Macmillan Company), Chennai, India(www.macmillansolutions.com)Printed in Malaysia by Vivar Printing Sdn Bhd

1 2009

v

Contents

Contributors, vii

Preface, xiii

Foreword, xv

PART I Concepts in Platelet Physiology, Function, and Measurement

1 Platelet physiology and the role of the platelet in ischemic heart disease, 3 Robert F. Storey

2 Laboratory assessment of platelet function and the effects of antiplatelet agents, 15 Alan D. Michelson, A.L. Frelinger, III

PART I I Pharmacology of Oral Antiplatelet Agents

3 Cyclooxygenase inhibitors, 33 Nina Chetty Raju, John W. Eikelboom

4 Aspirin response variability and resistance, 47 Wai-Hong Chen, Daniel I. Simon

5 P2Y12 inhibitors: Thienopyridines and direct oral inhibitors, 59 Jean-Philippe Collet, Boris Aleil, Christian Gachet, Gilles Montalescot

6 Thienopyridine response variability and resistance, 77 Udaya S. Tantry, Thomas A. Suarez, Paul A. Gurbel

PART I I I Pharmacology of Intravenous Antiplatelet Agents

7 Pharmacology of intravenous glycoprotein IIb/IIIa antagonists, 97 James C. Blankenship, Peter B. Berger

8 Intravenous P2Y12 inhibitors, 111 Steven P. Dunn, Steven R. Steinhubl

9 Antiplatelet effects of thrombin inhibitors and fi brinolytic agents, 125 Nicolai Mejevoi, Anjum Tanwir, Marc Cohen

PART IV Clinical Use of Antiplatelet Agents in Cardiovascular Disease

10 Antiplatelet therapy in acute coronary syndrome without ST elevation, 145 Christian W. Hamm

11 Antiplatelet therapy in ST-elevation myocardial infarction, 164 Michelle O’Donoghue, Marc S. Sabatine

12 Antiplatelet therapy in chronic coronary artery disease, 178 Kamran I. Muhammad, Deepak L. Bhatt

13 Antiplatelet therapy in peripheral arterial disease, 196 Esther S. Kim, Heather L. Gornik

14 Clinical use of antiplatelet agents in cardiovascular disease: cerebrovascular diseases, 215 Larry B. Goldstein

PART V Special Circumstances

15 Antiplatelet therapy and coronary bypass surgery: risks and benefi ts, 233 A. Burdess, N.L. Cruden, K.A. Fox

16 Management of antiplatelet therapy for non-cardiac surgery, 251 Nisheeth Goel, John F. Canales, James J. Ferguson

17 Antiplatelet therapy and coronary stents, 265 Alanna Coolong, Laura Mauri

Index, 281

Author Disclosure Table, 291

vi Contents

vii

Contributors

EditorStephen D. Wiviott, MDCardiovascular Division, Brigham and Women’s Hospital Assistant Professor of Medicine, Harvard Medical School Investigator, TIMI Study Group Boston, MA

Contributors

Boris Aleil, MDINSERM U311 Unit – Etablissement Français du Sang–Alsace (EFS-Alsace)StrasbourgFrance

Peter B. Berger, MDAssociate Chief Research Offi cerDirector, Center for Clinical StudiesGeisinger Health SystemsDanville, PA, USA

Deepak L. Bhatt, MD, FACC, FAHA Chief of Cardiology, VA Boston Healthcare SystemDirector, Integrated Interventional Cardiovascular Program at Brigham and Women’s Hospital and the VA Boston Healthcare SystemSenior Investigator, TIMI Study GroupBoston, MAUSA

James C. Blankenship, MDDirector, Cardiac Catheterization LaboratoriesGeisinger Medical CenterDanville, PA USA

Anne Burdess, BSc (Hons), MBCHB, MRCSClinical Research FellowDepartment of Clinical and Surgical SciencesUniversity of EdinburghEdinburghUK

John F. Canales, MDCardiology DepartmentTexas Heart InstituteBaylor College of MedicineSt. Luke’s Episcopal HospitalHouston, TxUSA

Wai-Hong Chen, MBBS, FACC, FAHADivision of Cardiology, Department of MedicineThe University of Hong KongQueen Mary HospitalHong Kong, China

Nina Chetty Raju, MBBS, FRACP, FRCPAClinical Research Fellow Thrombosis Service, Hamilton General HospitalDepartment of Medicine McMaster UniversityOntario, Canada

Marc Cohen, MD, FACCDirector, Division of CardiologyNewark Beth Israel Medical CenterNewark, NJUSA

Jean-Philippe Collet, MD, PhDINSERM U856 Unit – Institut de CardiologieCentre Hospitalier Universitaire Pitié-SalpêtrièreParisFrance

Alanna Coolong, MD, MScInstructor of MedicineBrigham and Women’s HopsitalHarvard Medical SchoolBoston, MAUSA

Dr Nicholas L. Cruden, PhD, MRCPClinical Lecturer in CardiologyUniversity of EdinburghEdinburghUK

viii Contributors

Steven P. Dunn, PharmDDepartment of Pharmacy Services and Division of CardiologyLinda and Jack Gill Heart InstituteUniversity of KentuckyLexington, KY, USA

John W. Eikelboom, MBBS, MSc, FRACP, FRCPAAssociate Professor Thrombosis Service, Hamilton General HospitalPopulation Health Research InstituteDepartment of Medicine, McMaster UniversityOntario, Canada

James J. Ferguson, MDCardiology ResearchSt. Luke’s Episcopal HospitalHouston, TXUSA

Keith A. Fox, BSC (Hons), FRCP, FESCBHF Professor of CardiologyUinveristy of EdinburghEdinburghUK

A.L. Frelinger, III, PhDAssociate Director, Center for Platelet Function StudiesAssociate Professor of Pediatrics and Molecular Genetics & MicrobiologyUniversity of Massachusetts Medical SchoolWorcester, MAUSA

Christian Gachet, MDINSERM U311 Unit – Etablissement Français du Sang–Alsace (EFS-Alsace)StrasbourgFrance

Nisheeth Goel, MDInterventional Cardiology FellowTexas Heart Institute/Baylor College of MedicineSt. Luke’s Episcopal HospitalHouston, TX, USA

Larry B. Goldstein, MD, FAAN, FAHADepartment of Medicine (Neurology)Center for Cerebrovascular DiseaseCenter for Clinical Health Policy ResearchDuke University and Durham VA Medical CenterDurham, NCUSA

Contributors ix

Heather L. Gornik, MD, MHSMedical Director, Non-Invasive Vascular LaboratoryDepartment of Cardiovascular MedicineAssistant Professor of MedicineCleveland ClinicLerner College of MedicineCleveland, OHUSA

Paul A. Gurbel, MDSinai Center for Thrombosis ResearchSinai HospitalBaltimore, MD, USA

Christian W. Hamm, MDMedical DirectorDirector of CardiologyKerckhoff Heart CenterBad NauheimGermany

Esther S. Kim, MD, MPHAssociate Staff PhysicianDepartment of Cardiovascular MedicineCleveland ClinicCleveland, OHUSA

Laura Mauri, MD, MScAssistant Professor of MedicineHarvard Medical SchoolDirector of Clinical BiometricsBrigham and Women’s HospitalBoston, MAUSA

Nicolai Mejevoi, MD, PhDFellow, Division of CardiologyNewark Beth Israel Medical Center Newark, NJUSA

Alan D. Michelson, MDDirector, Center for Platelet Function StudiesProfessor of Pediatrics, Medicine, and PathologyUniversity of Massachusetts Medical SchoolWorcester, MAUSA

Gilles Montalescot, MDINSERM U856 Unit – Institut de Cardiologie,Centre Hospitalier Universitaire Pitié-SalpêtrièreParisFrance

x Contributors

Kamran I. Muhammad, MDFellow in Cardiovascular MedicineCleveland ClinicCleveland, OH, USA

Michelle O’Donoghue, MDCardiovascular DivisionDepartment of MedicineMassachusetts General HospitalBoston, MA, USA

Marc S. Sabatine, MD, MPHCardiovascular DivisionTIMI Study GroupDepartment of MedicineBrigham and Women’s HospitalBoston, MA, USA

Daniel I. Simon, MDHarrington-McLanghlin Heart & Vascular InstituteUniversity Hospitals Case Medical CenterCase Western Reserve University School of MedicineCleveland, OH, USA

Steven R. Steinhubl, MDAssociate Professor of MedicineDivision of CardiologyLinda and Jack Gill Heart InstituteUniversity of KentuckyLexington, KY, USA

Robert F. Storey, BSc, BM, MRCP, DMCardiovascular Research UnitSchool of Medicine and Biomedical SciencesUniversity of Sheffi eld, UK

Thomas A. Suarez, MDSinai Center for Thrombosis ResearchSinai HospitalBaltimore, MD, USA

Udaya S. Tantry, PhDLaboratory DirectorSinai Center for Thrombosis ResearchSinai HospitalBaltimore, MD, USA

Anjum Tanwir, MDFellow, Division of CardiologyNewark Beth Israel Medical CenterNewark, NJ, USA

Contributors xi

Preface

Platelet activation and aggregation play key roles in the pathogenesis of atherosclerotic vascular disease. It is perhaps not surprising, then, that platelet function inhibitors have assumed a central role in the prevention of ischemic complications of these conditions including myocardial infarction, stroke, and limb ischemia and a major place in support of vascular procedures. In part because of the central role of these processes, there has been a growing iden-tifi cation of key pharmacologic targets and agents designed to alter these tar-gets in hopes of improving clinical outcomes. This book is therefore designed to assist the clinician, clinical and basic scientist, student, and allied health personnel in navigating this rapidly growing fi eld.

The book is divided into fi ve sections. First, concepts in platelet physiology, function, and measurement are introduced to provide a basic understand-ing of platelet biology and biometrics and serve as the foundation on which the rest of the text is built. The next two sections introduce the pharmacol-ogy of key oral and intravenous antiplatelet agents that are available to the clinician or in late stages of clinical development. These sections provide an understanding of mechanisms of action of these agents and their pharmacol-ogy leading to the sections on the clinical use of antiplatelet agents. The fourth section summarizes the use of antiplatelet therapy by atherosclerotic disease states including coronary artery disease (unstable angina and non-ST-elevation myocardial infarction, ST-elevation myocardial infarction, and chronic coronary artery disease), peripheral vascular disease, and cerebrovascular disease. The fi fth section, addresses “special circumstances” encountered by the clinician, the interaction between and management of antiplatelet agents in the settings of cardiac surgery, non-cardiac surgery, and percutaneous coronary intervention with stenting.

This book was made possible by an outstanding group of authors, all leaders in their fi elds, and their generous contributions are greatly appreciated.

xii

I would like to thank Dr. Elliott Antman, editor of this book series, who fi rst taught me how manage patients with ischemic vascular disease when I was a house offi cer, and later has served as an extremely generous research mentor. I would also like to thank Dr. Eugene Braunwald, the consummate physician scientist, for giving me an opportunity, and for giving all of us in academic medicine something to aspire to.

Stephen D. Wiviott, MD

Preface xiii

Foreword

The strategic driving force behind the American Heart Association’s mission of reducing disability and death from cardiovascular diseases and stroke is to change practice by providing information and solutions to healthcare pro-fessionals. The pillars of this strategy are Knowledge Discovery, Knowledge Processing, and Knowledge Transfer. The books in the AHA Clinical Series, of which Antiplatelet Therapy In Ischemic Heart Disease is included, focus on high-interest, cutting-edge topics in cardiovascular medicine. This book series is a critical tool that supports the AHA mission of promoting healthy behavior and improved care of patients. Cardiology is a rapidly changing fi eld and practi-tioners need data to guide their clinical decision-making. The AHA Clinical series, serves this need by providing the latest information on the physiology, diagnosis, and management of a broad spectrum of conditions encountered in daily practice.

Rose Marie Robertson, MD, FAHAChief Science Offi cer, American Heart Association

Elliott Antman, MD, FAHADirector, Samuel A. Levine Cardiac Unit,

Brigham and Women’s Hospital

xiv

PART I

Concepts in Platelet Physiology, Function, and Measurement

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3

Chapter 1

Platelet physiology and the roleof the platelet in ischemic heart diseaseRobert F. Storey

Blood platelets – equipped for action

The platelet, a tiny anucleate blood cell 2–4 µm in diameter, has major and diverse roles in health and disease and underlying this is a complex struc-ture that supports a wide range of functional responses [1]. Approximately 1 � 1011 new platelets are released each day into the circulation from bone marrow where they are formed by fragmentation from megakaryocytes [2,3]. Thrombopoietin is the most important cytokine regulating platelet produc-tion and some disease states, such as infl ammatory conditions, can increase thrombopoietin levels and platelet production [3]. This is of relevance when considering the rate of recovery of haemostatic function (and susceptibility to thrombosis) following exposure to irreversible platelet inhibitors such as aspi-rin and thienopyridines.

Platelets in the resting state are smooth, discoid cells possessing an open canalicular system and an exterior glycocalyx [1]. Ca2� is sequestered in intra-cellular stores and released into the cytoplasm upon activation of the plate-let by agonists, where it plays a major part in mediating platelet responses to activation [4]. Contained within the platelet cytoplasm are three types of granule, namely α-granules, dense granules and lysosomes [5]. α-Granules are the most abundant granules and contain a large number of proteins, many of which play a role in regulating the balance of thrombosis and fi brinolysis, such as α2-antiplasmin and plasminogen activator inhibitor-1 (PAI-1) [6]. The membranes of α-granules also contain proteins that are expressed on the cell surface following platelet activation, including glycoprotein (GP) IIb/IIIa

Antiplatelet Therapy in Ischemic Heart Disease, 1st edition. Edited by Stephen D. Wiviott.© 2009 American Heart Association, ISBN: 9-781-4051-7626-2

4 Part I Concepts in Platelet Physiology, Function, and Measurement

(αIIbβ3) and P-selectin (CD62P). Dense granules contain concentrated stores of ADP, ATP, pyrophosphate, ionized calcium and 5HT, and release of the con-tents of dense granules contributes to platelet activation and hemostasis [5,7]. Lysosomes contain numerous acid hydrolases and these are secreted by plate-lets only in response to strong agonist stimulation. Platelet activation leads to fusion of granules with the open canalicular system and release of granule contents [8].

Platelets possess a cytoskeleton which determines cell shape and consists mainly of microtubules and microfi laments [5]. Reorganization of the platelet cytoskeleton during platelet activation leads to a change in cell shape, with the platelets becoming spherical and extending fi nger-like projections known as pseudopodia. Activation of platelets also leads to a reorganization of components of the plasma membrane that leads to the assembly of prothrombinase complex on the platelet surface, catalyzing thrombin generation and coagulation [9].

Figure 1.1 provides an overview of mechanisms for platelet activation and associated responses which will be covered in subsequent sections.

The platelet glycoprotein IIb/IIIa complex

The GPIIb/IIIa complex is an adhesion receptor belonging to the integrin gene superfamily and, like other integrins, is a heterodimer composed of an α (αIIb) and a β (β3) transmembrane subunit [10]. Approximately 40,000 to 80,000 GPIIb/IIIa complexes are present on the surface of each resting platelet and this number can rapidly increase following activation by strong agonists due to exposure of internal receptors normally present within the open canal-icular system and α-granule membranes [10]. Activation of platelets induces conformational changes in GPIIb/IIIa so that it can bind fi brinogen, vWF or fi bronectin, whereas in the resting state GPIIb/IIIa binds fi brinogen only weakly so as to allow uptake into α-granules [11]. Fibrinogen molecules pos-sess two binding regions for GPIIb/IIIa and act as bivalent ligands, forming cross-bridges between activated platelets and leading to aggregation of acti-vated platelets. In this way, GPIIb/IIIa mediates the so-called “fi nal common pathway” of platelet aggregation, regardless of the stimulatory agent(s) [12]. The cytoplasmic domains of both IIb and IIIa play an important role in this “inside-out” signaling and also mediate “outside-in” signaling, whereby lig-and binding to GPIIb/IIIa leads to cytoskeletal reorganization and other post-ligand binding events that amplify platelet activation [13,14]. This explains why therapeutic concentrations of GPIIb/IIIa antagonists inhibit platelet dense granule secretion and platelet procoagulant responses as well as plate-let aggregation [15,16], although, through less well understood mechanisms, low concentrations of GPIIb/IIIa antagonists potentiate α-granule release and soluble CD40L release [17,18].

Fig. 1.1 Overview of platelet activation mechanisms and associated functional responses. Numerous agonists including thrombin, TXA2, collagen, 5HT, ADP and ATP bind to platelet surface receptors to initiate platelet activation and subsequent activation of GPIIb/IIIa (αIIbβ3) mediates platelet aggregation. Collagen binding to GPVI induces TXA2 formation and release. Dense granules release 5HT, ADP and ATP which amplify platelet activation, with activation of P2Y12 by ADP playing a major role in amplifi cation. α-Granule release promotes thrombus formation and associated infl ammatory responses. Microparticle formation and assembly of tenase and prothrombinase on the platelet surface promote thrombin generation. Nitric oxide (NO) and prostacyclin (PGI2) released by intact endothelium inhibit platelet activation. Adapted from Ref. [27] with permission.

Thrombin

TxA2

5HT

P2Y12

ADP ADPADP

5HT

Plateletactivation

P2Y15HT2A

PAR1

PAR4

Densegranule

Thrombingeneration

Shapechange

αIIbβ3

αIIbβ3αIIbβ3

Fibrinogen

Aggregation

Amplification

Coagulationfactors

In ammatory mediatorsβTG, PDGF, RANTES, PF4, TGFβ

TPα

Coagulation

GPVI

Collagen

ATPATP

P2X1

Aspirin

Clopidogrelprasugrel

Activemetabolite

AZD6140Cangrelor

GPIIB/IIIA antagonists

CD40LP-sel

sCD40L

Leukocyte bindingvia PSGL-1

Endothelium

Platelet-plateletinteraction

NO PGI2

_

Inhi

bitio

nPlatelet bindingvia αIIbβ3

α-granule

6 Part I Concepts in Platelet Physiology, Function, and Measurement

Platelet adhesion and initiation of thrombus formation

The presence on the platelet surface of various receptors that bind to adhesive ligands underlies the ability of platelets to adhere to subendothelial compo-nents that are exposed upon injury and breaching of the vascular endothe-lium, a process that represents the fi rst step in the hemostatic response of platelets [19]. Plasma von Willebrand factor (VWF) can bind to subendothelial components such as collagen, and platelets, via GPIbα in the GPIb-IX-V recep-tor complex, bind rapidly to immobilized VWF, providing a mechanism for platelet adhesion at high shear rates [19]. α2β1 and GPVI play pivotal roles in the binding of platelets to collagen in the subendothelial matrix, with GPVI playing a dominant role in the subsequent activation of platelets by colla-gen [19,20]. Initially, weak adhesion is stabilized through integrin activation and binding of VWF to GPIIb/IIIa as well as collagen to α2β1 [20]. Signaling through GPIb–IX–V, GPVI and GPIIb/IIIa leads to powerful activation of platelets and the release of soluble agonists via the following mechanisms: (1) release of dense granule contents containing the soluble agonists ADP, ATP and 5HT; (2) activation of phospholipase A2 and the formation and release of thromboxane A2; and (3) platelet procoagulant activity leading to generation of thrombin (Figure 1.1). These soluble agonists bind to platelet receptors that are linked to G proteins and mediate further platelet activation and recruit-ment of other platelets into platelet aggregates.

Platelet P2 receptors

There are three P2 receptor subtypes on the platelet surface, P2X1, P2Y1 and P2Y12 [21]. P2X1 is a ligand-gated cation channel activated by ATP and plays a role in platelet shape change and collagen-induced platelet activation [21]. P2Y1 and P2Y12 are G-protein coupled receptors activated by ADP. The P2Y1 receptor is linked to Gq and initiates ADP-induced platelet activation, this activation being then sustained and amplifi ed via P2Y12, which is coupled to Gi [21–24]. P2Y12 also plays a major role in sustaining and amplifying the responses to numerous agonists since other agonists induce dense granule release and ADP released from dense granules then binds to the P2Y recep-tors. In addition to sustaining and amplifying platelet aggregation, P2Y12 acti-vation amplifi es granule secretion and platelet procoagulant activity [24–26]. This is the basis for the importance of P2Y12 in platelet function and the grow-ing therapeutic success of antagonists that target this receptor [27].

In a similar fashion to ADP’s action via the P2Y12 receptor, epinephrine (adrenaline) and norepinephrine (noradrenaline) can also amplify platelet activation via α2A adrenergic receptors but the observation that this requires supraphysiological concentrations of epinephrine and norepinephrine renders the pathophysiological signifi cance of this pathway uncertain [28].

Chapter 1 Platelet physiology 7

Platelet protease-activated receptors (PARs) and thrombin

PAR1 and PAR4 are expressed on human platelets with PAR1 likely playing the more important role, since it is activated at low thrombin concentrations whereas PAR4 requires higher thrombin concentrations in order to contribute to thrombin-induced platelet responses [29]. Thrombin cleaves the N-terminal exodomain on the PARs leading to a tethered peptide ligand that activates the receptor, a process that can be mimicked by thrombin receptor-activating pep-tides or TRAPs [29]. Whereas the P2Y1 receptor is linked only to Gq, PAR1 is linked to both Gq and G12/13 and mediates strong platelet activation as man-ifest by the extent of granule secretion and procoagulant activity induced by PAR1 activation [29–31]. P2Y12 activation plays a key role in promoting these responses [24,26,32]. GPIbα may serve as a cofactor at the platelet surface, sup-porting PAR cleavage by thrombin [29,33]. The presence of PAR1 on other cell types involved in infl ammatory responses provides a rationale for targeting this receptor in order to treat thrombotic diseases and associated infl ammation [29].

The arachidonic acid pathway and thromboxane A2

A group of phospholipases, collectively termed phospholipase A2 (PLA2), hydro-lyse membrane phospholipids, such as phosphatidylcholine and phosphatidyl-serine, to produce arachidonic acid [34,35]. Arachidonic acid is rapidly converted by cyclooxygenase (COX) to prostaglandin G2, which is then converted by per-oxidase to prostaglandin H2 (PGH2) [34,36]. PGH2 is then rapidly converted by thromboxane synthase to thromboxane A2 (TXA2). PGH2 and TXA2 are highly labile, potent platelet agonists that can diffuse across the plasma membrane and bind to specifi c G-protein coupled platelet receptors [30,34,37]. Studies of aspirin (acetylsalicylic acid), which acetylates and irreversibly inhibits COX, demonstrate the role of the arachidonic acid pathway in platelet responses to stimulation by different agonists. Aspirin abolishes platelet macroaggregation induced by ara-chidonic acid and substantially reduces platelet aggregation induced by low concentrations of collagen [38,39]. It also inhibits the “secondary wave” of macro-aggregation induced by ADP, adrenaline and platelet-activating factor in citrated platelet-rich plasma but has little or no effect on platelet aggregation induced by these agonists in media containing physiological levels of divalent cations, indi-cating a restricted role for the arachidonic acid pathway in platelet activation under physiological conditions [24,40–43]. This explains why effective inhibition of COX by aspirin leaves many aspects of platelet function relatively intact.

Receptor pathways that inhibit platelet activation

The vascular endothelium presents an antithrombotic surface, in part related to the release by intact endothelium of nitric oxide (NO) and prostacyclin

8 Part I Concepts in Platelet Physiology, Function, and Measurement

(PGI2), both of which act on platelet pathways that suppress platelet activa-tion as well as having vasodilatory effects [44]. NO activates platelet guany-lyl cyclase, raising platelet cyclic GMP levels and inhibiting agonist-induced rises in cytoplasmic calcium levels [45,46]. Platelet-derived NO also appears to limit thrombus formation and synthetic NO donors may have substantial antithrombotic effects [46,47]. Glutathione peroxidase potentiates inhibition of platelets by NO donors and inherited defi ciency of the plasma isoform of this enzyme can lead to childhood ischemic stroke [46].

Endothelial COX-1 and prostaglandin G/H synthase-2 (PGHS-2; known as COX-2) mediate the production of PGI2, which activates IP receptors on plate-lets and, via coupling to Gs, mediates an increase in platelet cyclic AMP, which in turn inhibits platelet activation [48,49]. The inhibition of these endothelial COX enzymes by traditional non-steroidal anti-infl ammatory drugs (NSAIDs) and newer selective COX-2 inhibitors, and subsequent impairment of endothe-lial PGI2 release, underlies the adverse cardiovascular effects of these drugs [49].

Another COX metabolite, PGE2, has contradictory actions on platelets but, acting at low concentration via EP3 receptors, may enhance platelet responses by opposing increases in cyclic AMP [50]. It is suggested that release of PGE2 from infl amed vessel wall, such as atherosclerotic plaque, may counteract the inhibitory effects of PGI2 and contribute to arterial thrombosis [50,51].

Adenosine has an inhibitory effect on platelet function, acting via A2A recep-tors and increasing cyclic AMP levels [52]. It is proposed that plasma levels of adenosine increase suffi ciently during ischemia or hypoxia to activate these receptors [52]. Adenosine and other A2A receptor agonists have antithrombotic effects in animal models of thrombosis [53]. The antiplatelet drug dipyrida-mole acts by inhibiting adenosine uptake by blood cells, thereby increasing exposure of platelets to adenosine, as well as by inhibiting platelet cyclic GMP-dependent phosphodiesterase [54].

Platelet procoagulant activity

In their resting state, platelets have asymmetric distribution of aminophos-pholipids in the surface membrane bilayer with enzymes acting to keep these aminophospholipids (predominantly phosphatidylserine and phosphati-dylethanolamine) in the inner layer [55]. Platelet activation is associated with an increase in the cytoplasmic ionized calcium concentration that inhibits the activity of these enzymes and also activates the enzyme scramblase that causes redistribution of the aminophospholipids to the outer layer where they are able to support the assembly of tenase and prothrombinase complexes and subsequent generation of thrombin in plasma. Microparticles are also shed under the action of calpain and these also have procoagulant properties. These processes are triggered by platelet GPVI receptor binding to collagen and play an important role in thrombin generation and arterial thrombogenesis [56].

Chapter 1 Platelet physiology 9

Thrombin-induced activation of platelets further promotes platelet procoagu-lant activity, and P2Y12 receptor activation and GPIIb/IIIa receptor outside-in signaling play important roles in amplifying this process, such that antagonists of these receptors inhibit platelet procoagulant responses [16,24,32,57–59].

Infl ammatory responses of platelets

α-Granule secretion consequent to platelet activation leads to a number of processes that are considered pro-infl ammatory. CD40L and P-selectin are translocated to the platelet surface as a result of fusing of the α-granule mem-brane with the surface membrane and are then gradually shed into the sur-rounding medium in their soluble forms. Soluble CD40L (sCD40L) induces cytokine production in vascular cells and may play a role in atherogenesis and restenosis, as well as promoting thrombosis by stabilizing platelet aggregates [60,61]. P-selectin binds to its counter-receptor on leukocytes, PSGL-1, help-ing to recruit leukocytes and monocyte-derived microparticles into thrombus, and the ensuing cross-talk between platelets, leukocytes and the microparti-cles promotes thrombogenesis and infl ammatory responses [62,63]. Release of the chemokine RANTES by platelets, in conjunction with P-selectin, supports the binding of monocytes to infl amed endothelium and contributes to intimal hyperplasia in murine models [64,65]. β-thromboglobulin, platelet-derived growth factor and platelet factor 4 are other platelet α-granule contents that also contribute to infl ammatory responses [6].

Nucleotides released from platelet dense granules may also have pro-infl ammatory effects: both ADP and ATP can activate leukocytes whilst ATP acts on P2X receptors on vascular smooth muscle cell (VSMC) and may con-tribute to VSMC proliferation and migration [66,67]. These nucleotides also act on endothelial cells to promote nitric oxide and prostacyclin release [66].

The role of the platelet in coronary atherothrombosis

Atherothrombosis refers to the process whereby progression of atherosclerosis leads to plaque rupture or erosion, which induces arterial thrombus formation and, in some instances, clinical sequelae such as coronary artery thrombosis causing acute coronary syndromes or sudden cardiac death [68,69]. Coronary arterial plaques that have a thin fi brous cap overlying a lipid-rich core and abundance of infl ammatory cells are particularly prone to rupture and are termed “vulnerable” or “high-risk” plaques [68,70]. Exposure of collagen, vWF and fi bronectin following endothelial disruption leads to platelet adhe-sion and activation, as described in the previous section but, furthermore, the exposed lipid-rich core of high-risk plaque is highly thrombogenic, contain-ing abundant tissue factor that initiates the coagulation cascade culminat-ing in thrombin formation, which then leads to platelet activation and fi brin

10 Part I Concepts in Platelet Physiology, Function, and Measurement

deposition [68,69]. The central role of thrombin in the thrombosis that ensues following plaque disruption explains why anticoagulants such as heparins, direct thrombin inhibitors and factor Xa inhibitors have benefi cial effects in the management of acute coronary syndromes [71–73] in addition to antiplate-let agents that target pathways that are involved in the platelet responses to thrombin and collagen, as described above. As well as exposure of tissue factor in the vessel wall, there is a circulating pool of tissue factor in blood that becomes concentrated at the site of vessel wall injury and contributes to thrombus formation [74]. Platelets, leukocytes and endothelial cells can express tissue factor, which may then be borne on circulating microparticles derived from these cells [75].

The formation of non-occlusive, platelet-rich thrombi over the sites of athero-sclerotic plaque erosion or rupture with subsequent release of infl ammatory mediators from platelet α-granules and platelet-leukocyte interactions may con-tribute to the progression of atherosclerotic lesions [76]. Such mural thrombi in coronary arteries may not be immediately associated with any clinical manifes-tations but instead contribute to the progressive narrowing of the arterial lumen that eventually leads to myocardial ischemia under conditions of increased myocardial oxygen demand and the associated symptom of angina pectoris. Platelets may also contribute to the vascular smooth muscle cell proliferation that leads to restenosis following percutaneous coronary intervention [77].

Conclusion

The rich variety of characteristics of platelets equips them for their role in hemostasis and physiological responses to vascular injury. Except in excep-tional circumstances, it is only when the vessel wall becomes diseased that these physiological responses tend to be exaggerated and lead to thrombotic occlusion of the vessel lumen and excessive infl ammation of the vessel wall. This explains why the platelet plays such an important role in the various manifestations of ischemic heart disease. Advances in the knowledge of plate-let physiology have led to the development of antithrombotic therapies for managing ischemic heart disease and continue to inform the development of novel strategies.

References 1 White JG. Anatomy and structural organization of the platelet. In: Colman RW,

Hirsh J, Marder VJ, Salzman EW, eds. Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd edn. J.B. Lippincott Company, Philadelphia, 1994: 397–413.

2 Chang Y, Bluteau D, Deb Il I N, Vainchenker W. From hemopoietic stem cells to platelets. J Thromb Haemost 2007; 5 (Suppl. 1): 318–327.

3 Deutsch VR, Tomer A. Megakaryocyte development and platelet production. Br J Haematol 2006; 134: 453–466.

Chapter 1 Platelet physiology 11

4 Rosado JA, Sage SO. A role for the actin cytoskeleton in the initiation and mainte-nance of store-mediated calcium entry in human platelets. Trends Pharmacol Sci 2000; 10: 327–332.

5 Lind SE. Platelet morphology. In: Loscalzo J, Schafer AI, eds. Thrombosis and Hemorrhage. Blackwell Scientifi c Publications, Boston, 1994: 201–218.

6 Harrison P, Cramer EM. Platelet alpha-granules. Blood Rev 1993; 7: 52–62. 7 McNicol A, Israels SJ. Platelet dense granules: structure, function and implications

for haemostasis. Thromb Res 1999; 95: 1–18. 8 White JG, Krumwiede M. Further studies of the secretory pathway in thrombin-

stimulated human platelets. Blood 1987; 69: 1196–1203. 9 Walsh PN. Platelet-coagulant protein interactions. In: Colman RW, Hirsh J,

Marder VJ, Salzman EW, eds. Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd edn. J.B. Lippincott Company, Philadelphia 1994: 629–651.

10 Shattil SJ. Function and regulation of the B3 integrins in hemostasis and vascular biology. Thromb Haemost 1995; 74: 149–155.

11 Clemetson KJ. Platelet activation: signal transduction via membrane receptors. Thromb Haemost 1995; 74: 111–116.

12 Charo IF, Kieffer N, Phillips DR. Platelet membrane glycoproteins. In: Colman RW, Hirsh J, Marder VJ, Salzman EW, eds. Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd edn. Philadelphia: Lippincott Company 1994: 603–628.

13 Levy-Toledano S, Gallet C, Nadal F, Bryckaert M, Maclouf J, Rosa J-P. Phosphorylation and dephosphorylation mechanisms in platelet function: a tightly regulated balance. Thromb Haemost 1997; 78: 226–233.

14 Shattil SJ, Gao J, Kashiwagi H. Not just another pretty face: regulation of platelet function at the cytoplasmic face of integrin αIIbB3. Thromb Haemost 1997; 78: 220–225.

15 Storey RF. The platelet P2Y12 receptor as a therapeutic target in cardiovascular dis-ease. Platelets 2001; 12: 197–209.

16 Judge HM, Buckland RJ, Holgate CE, Storey RF. Glycoprotein IIb/IIIa and P2Y12 receptor antagonists yield additive inhibition of platelet aggregation, granule secretion, soluble CD40L release and procoagulant responses. Platelets 2005; 16: 398–407.

17 Schneider DJ, Taatjes DJ, Sobel BE. Paradoxical inhibition of fi brinogen binding and potentiation of alpha-granule release by specifi c types of inhibitors of glycoprotein IIb-IIIa. Cardiovascular Research 2000; 45: 437–446.

18 Nannizzi-Alaimo L, Alves VL, Phillips DR. Inhibitory effects of glycoprotein IIb/IIIa antagonists and aspirin on the release of soluble CD40 ligand during platelet stimu-lation. Circulation 2003; 107: 1123–1128.

19 Ruggeri ZM, Mendolicchio GL. Adhesion mechanisms in platelet function. Circ Res 2007; 100: 1673–1685.

20 Watson SP, Auger JM, McCarty OJT, Pearce AC. GPVI and integrin αIIbβ3 signaling in platelets. J Thromb Haemost 2005; 3: 1752–1762.

21 Hechler B, Cattaneo M, Gachet C. The P2 receptors in platelet function. Sem Thromb Haemost 2005; 31: 150–161.

22 Jin J, Kunapuli SP. Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation. Proc Natl Acad Sci USA 1998; 95: 8070–8074.

23 Hechler B, Leon C, Vial C, Vigne P, Frelin C, Cazenave JP, et al. The P2Y1 receptor is necessary for adenosine-5’-diphosphate-induced platelet aggregation. Blood 1998; 92: 152–159.

12 Part I Concepts in Platelet Physiology, Function, and Measurement

24 Storey RF, Sanderson HM, White AE, May JA, Cameron KE, Heptinstall S. The cen-tral role of the P2T receptor in amplifi cation of human platelet activation, aggrega-tion, secretion and procoagulant activity. Br J Haematol 2000; 110: 925–934.

25 Cattaneo M, Lombardi R, Zighetti ML, Gachet C, Ohlmann P, Cazenave J-P, et al. Defi ciency of (33P)2MeS-ADP binding sites on platelets with secretion defect, normal granule stores and normal thromboxane A2 production: evidence that ADP poten-tiates platelet secretion independently of the formation of large platelet aggregates and thromboxane A2 production. Thromb Haemost 1997; 77: 986–990.

26 Trumel C, Payrastre B, Plantavid M, Hechler B, Vial C, Presek P, et al. A key role of adenosine diphosphate in the irreversible platelet aggregation induced by the PAR-1 activating peptide through the late activation of phosphoinositide 3-kinase. Blood 1999; 94: 4156–4165.

27 Storey RF. Biology and pharmacology of the platelet P2Y12 receptor. Curr Pharm Design 2006; 12: 1255–1259.

28 Colman RW, Cook JJ, Niewiarowski S. Mechanisms of platelet aggregation. In: Colman RW, Hirsh J, Marder VJ, Salzman EW, eds. Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd edn. Lippincott Company, Philadelphia 1994: 508–523.

29 Coughlin SR. Protease-activated receptors in hemostasis, thrombosis and vascular biology. J Thromb Haemost 2005; 3: 1800–1814.

30 Offermanns S, Laugwitz K, Spicher K, Schultz G. G Proteins of the G12 family are activated via thromboxane A2 and thrombin receptors in human platelets. Proc Natl Acad Sci USA 1994; 91: 504–508.

31 Offermanns S, Toombs CF, Hu Y-H, Simon MI. Defective platelet activation in Gαq-defi cient mice. Nature 1997; 389: 183–186.

32 Dorsam RT, Tuluc M, Kunapuli SP. Role of protease-activated and ADP receptor sub-types in thrombin generation on human platelets. J Thromb Haemost 2004; 2: 804–812.

33 Celikel R, McClintock RA, Roberts JR, Mendolicchio GL, Ware J, Varughese KI, et al. Modulation of alpha-thrombin function by distinct interactions with platelet glyco-protein Ibalpha. Science 2003; 301: 218–221.

34 Moncada S, Vane JR. Arachidonic acid metabolites and the interactions between platelets and blood vessel walls. New Engl J Med 1979; 300: 1142–1147.

35 Rittenhouse-Simmons S. Differential activation of platelet phospholipases by thrombin and ionophore A23187. J Biol Chem 1981; 256: 4153–4155.

36 Lagarde M. Metabolism of fatty acids by platelets and the functions of various metabolites in mediating platelet function. Prog Lipid Res 1988; 27: 135–152.

37 Brass LF, Hoxie JA, Manning DR. Signaling through G proteins and G protein- coupled receptors during platelet activation. Thromb Haemost 1993; 70: 217–223.

38 Kuster LJ, Frolich JC. Platelet aggregation and thromboxane release induced by ara-chidonic acid, collagen, ADP and platelet-activating factor following low dose ace-tylsalicylic acid in man. Prostaglandins 1986; 32: 415–423.

39 Berglund U, Wallentin L. Persistent inhibition of platelet function during long-term treatment with 75 mg acetylsalicylic acid daily in men with unstable coronary artery disease. Eur Heart J 1991; 12: 428–433.

40 Heptinstall S, Mulley GP. Adenosine diphosphate-induced platelet aggregation and release reaction in heparinized platelet rich plasma and the infl uence of added cit-rate. Br J Haematol 1977; 36: 565–571.

Chapter 1 Platelet physiology 13

41 Heptinstall S, Taylor PM. The effects of citrate and extracellular calcium ions on the platelet release reaction induced by adenosine diphosphate and collagen. Thromb Haemost 1979; 42: 778–793.

42 Lages B, Weiss HJ. Dependence of human platelet functional responses on divalent cations: aggregation and secretion in heparin- and hirudin-anticoagulated platelet-rich plasma and the effects of chelating agents. Thromb Haemost 1981; 45: 173–179.

43 Packham MA, Bryant NL, Guccione MA, Kinlough-Rathbone RL, Mustard JF. Effect of concentration of Ca2� in the suspending medium on the responses of human and rabbit platelets to aggregating agents. Thromb Haemost 1989; 62: 968–976.

44 Cines DB, Pollak ES, Buck CA, Loscalzo J, Zimmerman GA, McEver RP, et al. Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 1998; 91: 3527–3561.

45 Mendelsohn ME, O’Neill S, George D, Loscalzo J. Inhibition of fi brinogen binding to human platelets by S-nitroso-N-acetylcysteine. J Biol Chem 1990; 265: 19028–19034.

46 Loscalzo J. Nitric oxide insuffi ciency, platelet activation, and arterial thrombosis. Circ Res 2001; 88: 756–762.

47 Vilahur G, Segales E, Salas E, Badimon L. Effects of a novel platelet nitric oxide donor (LA816), aspirin, clopidogrel, and combined therapy in inhibiting fl ow- and lesion-dependent thrombosis in the porcine ex vivo model. Circulation 2004; 110: 1686–1693.

48 Boie Y, Rushmore TH, Darmon-Goodwin A, Grygorczyk R, Slipetz DM, Metters KM, et al. Cloning and expression of a cDNA for the human prostanoid IP receptor. J Biol Chem 1994; 269: 12173–12178.

49 Grosser T, Fries S, FitzGerald GA. Biological basis for the cardiovascular conse-quences of COX-2 inhibition: therapeutic challenges and opportunities. J Clin Invest 2006; 116: 4–15.

50 Fabre J-E, Nguyen M, Athirakul K, Coggins K, McNeish JD, Austin S, et al. Activation of the murine EP3 receptor for PGE2 inhibits cAMP production and promotes plate-let aggregation. J Clin Invest 2001; 107: 603–610.

51 Gross S, Tilly P, Hentsch D, Vonesch J-L, Fabre J-E. Vascular wall-produced prostag-landin E2 exacerbates arterial thrombosis and atherothrombosis through platelet EP3 receptors. J Exp Med 2007; 204: 311–320.

52 Gessi S, Varani K, Merighi S, Ongini E, Borea PA. A2A adenosine receptors in human peripheral blood cells. Br J Pharmacol 2000; 129: 2–11.

53 Linden MD, Barnard MR, Frelinger AL, Michelson AD, Przyklenk K. Effect of adeno-sine A2 receptor stimulation on platelet activation-aggregation: Differences between canine and human models. Thromb Res 2008; 121: 689–698.

54 Schaper W. Dipyridamole, an underestimated vascular protective drug. Cardiovasc Drug Ther 2005; 19: 357–363.

55 Solum NO. Procoagulant expression in platelets and defects leading to clinical disor-ders. Arterioscler Thromb Vasc Biol 1999; 19: 2841–2846.

56 Siljander P, Farndale RW, Feijge MAH, Comfurius P, Kos S, Bevers EM, et al. Platelet adhesion enhances the glycoprotein VI-dependent procoagulant response: involve-ment of p38 MAP kinase and calpain. Arterioscler Thromb Vasc Biol 2001; 21: 618–627.

57 Keularts IM, Beguin S, de Zwaan C, Hemker HC. Treatment with a GPIIb/IIIa antag-onist inhibits thrombin generation in platelet rich plasma from patients. Thromb Haemost 1998; 80: 370–371.

14 Part I Concepts in Platelet Physiology, Function, and Measurement

58 Ramstrom S, Ranby M, Lindahl TL. Effects of inhibition of P2Y1 and P2Y12 on whole blood clotting, coagulum elasticity and fi brinolysis resistance studied with free oscil-lation rheometry. Thromb Res 2003; 109: 315–322.

59 Keuren JF, Wielders SJH, Ulrichts H, Hackeng T, Heemskerk JWM, Deckmyn H, et al. Synergistic effect of thrombin on collagen-induced platelet procoagulant activ-ity is mediated through protease-activated receptor-1. Arterioscler Thromb Vasc Biol 2005; 25: 1499–1505.

60 Henn V, Slupsky JR, Grafe M, Anagnostopoulos I, Forster R, Muller-Berghaus G, et al. CD40 ligand on activated platelets triggers an infl ammatory reaction of endothelial cells. Nature 1998; 391: 591–594.

61 Andre P, Nannizzi-Alaimo L, Prasad SK, Phillips DR. Platelet-derived CD40L: the switch-hitting player of cardiovascular disease. Circulation 2002; 106: 896–899.

62 Furie B, Furie BC, Flaumenhaft R. A journey with platelet P-selectin: the molecular basis of granule secretion, signalling and cell adhesion. Thromb Haemost 2001; 86: 214–221.

63 Andre P. P-selectin in haemostasis. Br J Haematol 2004; 126: 298–306.64 von Hundelshausen P, Weber KSC, Huo Y, Proudfoot AEI, Nelson PJ, Ley K, et al.

RANTES deposition by platelets triggers monocyte arrest on infl amed and athero-sclerotic endothelium. Circulation 2001; 103: 1772–1777.

65 Schober A, Manka D, von Hundelshausen P, Huo Y, Hanrath P, Sarembock IJ, et al. Deposition of platelet RANTES triggering monocyte recruitment requires P- selectin and is involved in neointima formation after arterial injury. Circulation 2002; 106: 1523–1529.

66 Kunapuli SP, Daniel JL. P2 receptor subtypes in the cardiovascular system. Biochem J 1998; 336: 513–523.

67 Erlinge D. Extracellular ATP: a growth factor for vascular smooth muscle cells. Gen Pharmacol 1998; 31: 1–8.

68 Viles-Gonzalez JF, Fuster V, Badimon JJ. Atherothrombosis: A widespread dis-ease with unpredictable and life-threatening consequences. Eur Heart J 2004; 25: 1197–1207.

69 Ruggeri ZM. Platelets in atherothrombosis. Nature Medicine 2002; 8: 1227–1234.70 Robbie L, Libby P. Infl ammation and atherothrombosis. Ann N Y Acad Sci 2001; 947:

167–180.71 Wallentin L. Prevention of cardiovascular events after acute coronary syndrome.

Semin Vasc Med 2005; 5: 293–300.72 Antman EM, Cohen M. Newer antithrombin agents in acute coronary syndromes.

Am Heart J 1999; 138: S563–569.73 Oasis-6 Trial Group. Effects of fondaparinux on mortality and reinfarction in patients

with acute ST-segment elevation myocardial infarction: The OASIS-6 randomized trial. JAMA 2006; 295: 1519–1530.

74 Balasubramanian V, Grabowski E, Bini A, Nemerson Y. Platelets, circulating tissue fac-tor, and fi brin colocalize in ex vivo thrombi: real-time fl uorescence images of thrombus formation and propagation under defi ned fl ow conditions. Blood 2002; 100: 2787–2792.

75 Mackman N, Tilley RE, Key NS. Role of the extrinsic pathway of blood coagulation in hemostasis and thrombosis. Arterioscler Thromb Vasc Biol 2007; 27: 1687–1693.

76 Libby P. Changing concepts of atherogenesis. J Int Med 2000; 247: 349–358.77 Weber A-A, Schror K. The signifi cance of platelet-derived growth factors for

proliferation of vascular smooth muscle cells. Platelets 1999; 10: 77–96.

15

Chapter 2

Laboratory assessment ofplatelet function and the effectsof antiplatelet agentsAlan D. Michelson and A.L. Frelinger, III

Introduction

Because platelets have a central role in ischemic heart disease [1,2] (as dis-cussed in detail in Chapter 1 of this book), antiplatelet therapy has been demonstrated to be benefi cial in this clinical setting [3,4]. However, there is variability between patients in the response of their platelets to antiplatelet therapy [5]. There is therefore increasing interest in the use of platelet func-tion tests to monitor the effects of antiplatelet drugs in ischemic heart disease, with the goal of guiding antiplatelet therapy to the optimal dose for preven-tion or treatment of thrombosis while minimizing hemorrhagic side effects [5]. These platelet function tests are frequently used for the measurement of “aspirin resistance” or “clopidogrel resistance” [5–7]. The clinical relevance of “ resistance”, also referred to as response variability, is discussed in detail in Chapters 4 and 6 of this book. The present chapter reviews the laboratory assessment of platelet function and the effects of antiplatelet agents. Tables 2.1–2.3 summarize laboratory methods that can potentially be used to guide the clinical use of antiplatelet drugs in ischemic heart disease.

The bleeding time

The bleeding time, the fi rst test of platelet function, was developed in the early 1900s [8]. The basis of the test is the timed, platelet-dependent cessation of bleed-ing from a standardized in vivo wound. Although the bleeding time is therefore a physiologically relevant test, it has many disadvantages: non-specifi city (e.g. affected by von Willebrand factor), insensitivity, high interoperator variability

Antiplatelet Therapy in Ischemic Heart Disease, 1st edition. Edited by Stephen D. Wiviott.© 2009 American Heart Association, ISBN: 9-781-4051-7626-2

16 Part I Concepts in Platelet Physiology, Function, and M

easurement

Table 2.1 Methods for the laboratory assessment of platelet function.

Test Basis Advantages Disadvantages

Turbidometric aggregometry Platelet aggregation Historical gold standard High sample volumeSample preparationTime consuming

Impedance aggregometry Platelet aggregation Whole blood assay High sample volumeSample preparationTime consuming

VerifyNow® Platelet aggregation Simple, rapidPoint-of-care (no pipetting required)Low sample volumeWhole blood assay

Limited hematocrit & platelet count range

Plateletworks® Platelet aggregation Minimal sample preparationWhole blood assay

Not well studied

TEG® Platelet Mapping™ system Platelet contribution to clot strength

Whole blood assayClot information

Limited studiesRequires pipetting

Impact® cone-and-plate(let) analyzer

Shear induced platelet adhesion

Simple, rapidPoint-of-careLow sample volumeNo sample preparationWhole blood assayShear-dependent

Instrument not widely availableRequires pipeting